Physiological roles of CIDEs CIDEA deficient mice exhibit lean phenotype and are obesity resistant 6

Chapter Chapter Discussion 106 Chapter The original objective of this work was to investigate the physiological role of Cidea, a molecule presumably related to apoptosis. Cidea had been shown originally as an apoptotic effector when ectopically overexpressed in cell lines (Inohara et al., 1998). It was therefore somewhat surprising to discover that Cidea is expressed almost exclusively in BAT. Cidea null mice show hyperactive BAT, with a higher basic metabolic rate and more active lipid metabolism. Furthermore, BAT of Cidea-null mice had enhanced lipolytic activities. Cidea null mice have a hyperactive thermogenesis and increased lipolysis in response to cold and with increasing age compared to wild type mice. Notably, Cidea null mice were lean, with enhanced glucose disposal and resistance to high fat diet induced obesity and diabetes. Less fat accumulation coupled with enhanced glucose disposal in Cidea-null mice is probably due to the higher energy expenditure in their BAT. One possible molecular mechanism underlying these phenomena, the direct inhibition of UCP1 activity by Cidea was further revealed by biochemical and cellular investigations. Defects in adaptive thermogenesis in BAT have been proposed to be a critical physiological defense mechanism against obesity and diabetes (Hamann et al., 1998). Nearly all experimental rodent models of obesity are accompanied by diminished or defective BAT function (Cui et al., 1990). Disrupting BAT function by denervation or excision of interscapular BAT increases mouse body weight (Dulloo and Miller, 1984). Deletion of the cAMP dependent PKA regulatory subunit II β (PKA RII β), which is abundantly expressed in BAT, WAT and brain, results in elevated body temperature, higher basal metabolic rate and a leaner phenotype (Cummings et al., 1996). Transgenic mice with BAT disrupted by overexpression of diphtheria toxin in brown adipocytes were obese with symptoms of hyperglycemia and insulin resistance 107 Chapter (Lowell et al., 1993). In addition, alteration of the uncoupling activity in transgenic mice that ectopically express UCP1 in WAT (Chen and Farese, 2001; Kopecky et al., 1995; Kopecky et al., 1996a) or UCP3 in skeletal muscle results in resistance to dietinduced obesity and diabetes (Clapham et al., 2000). Mice lacking all three β- adrenergic receptors have reduced metabolic rates due to the lack of diet-induced thermogenesis and developed obesity when fed a high fat diet (Bachman et al., 2002). Despite the strong correlation between BAT with obesity, direct evidence of obesity resulting from BAT specific genes is scarce. Although BAT and WAT exert opposite functions in terms of energy expenditure, most genes involved in adipocyte differentiation and lipid metabolism are expressed in both tissues. UCP1 is the only gene identified thus far that is expressed specifically in BAT but not in WAT. Mice deficient in UCP1 showed no weight gain, which may be due to compensatory effects by other uncoupling proteins (Enerback et al., 1997). The adipose tissue has emerged as an endocrine organ that is central to the regulation of energy homeostasis. It can secrete proteins that exert a pleiotropic effect in response to nutritional changes. These proteins are involved in glucose and fat metabolism and hence can influence insulin resistance. They include leptin (Friedman and Halaas, 1998), resistin (Steppan et al., 2001), adiponectin (Arita et al., 1999), (Cianflone et al., 1989), tumour necrosis factor-alpha (Sewter et al., 1999) and interleukin-6 (Mohamed-Ali et al., 1997). The identification of the mutant gene underlying the obese phenotype of the ob/ob mouse was made by Zhang et al. using positional cloning, and had led to the characterisation of the hormone leptin (Zhang et al., 1994). Initial work indicated that the leptin gene is expressed only in WAT, but subsequent findings have shown it to be expressed at lower levels in other forms of adipose tissue, including BAT. A variety of other tissues (e.g. bone, mammary gland, 108 Chapter ovarian follicles, the placenta, stomach and certain fetal organs, such as the heart and bone) have now been shown to contain leptin and the leptin gene can be induced in muscle (Chelikani et al., 2003; Friedman and Halaas, 1998). The placenta is a site of leptin synthesis in humans, rodents and ruminants (Hoggard et al., 1997; Masuzaki et al., 1997; Senaris et al., 1997). Nevertheless, leptin is secreted primarily from WAT as an indicator of the level of fat storage in the body and stimulates long-form Ob-rb receptors in the hypothalamus to decrease food intake and increase energy expenditure (Friedman and Halaas, 1998). Apart from the few instances where leptin is absent, leptin levels are generally increased in obesity, while the sympathetic sensitivity of the adipose tissue is reduced. The dysregulation of energy balance leading to obesity may partly involve a decrease in leptin sensitivity, or the leptin system may be set to have maximal effects at low leptin levels. Although their leptin levels were lower, Cideanull mice did not show a greater food uptake, indicating that they might be hypersensitive to leptin. A similar phenotype has been reported in mice lacking the translational inhibitor 4e-bp1 (Tsukiyama-Kohara et al., 2001). Cidea may therefore be part of a regulatory feedback pathway involving the central nervous system and WAT. It could even be a regulator of the leptin-signaling cascade. Crossing the ob/ob mice with Cidea null mice would be a future line of research that could resolve this possibility. The thermogenic role of UCP1 has been definitively proven by genetic deletion of the UCP1 gene. Mice lacking UCP1 showed a rapid decrease in core body temperature during cold exposure (Cassard-Doulcier et al., 1998; Enerback et al., 1997). The uncoupling activity of UCP1 and other uncoupling proteins has been studied extensively in yeast in which the expression of the proteins of interest can be tightly controlled (Bouillaud et al., 1994; Klingenberg and Echtay, 2001). Exposure of 109 Chapter animals to cold or stimulation by pharmacological agents such as norepinephrine results in β-adrenergic receptor activation, elevation of intracellular cAMP and activation of cAMP dependent protein kinase A (PKA) in BAT (Hagen and Lowell, 2000). Lipid hydrolysis and UCP1 activity are dramatically increased in response to the elevation of cAMP and the activation of PKA. Free fatty acids (FFA) serve both as an energy substrate for the respiratory chain as well as an activator of UCP1 to enhance thermogenesis. Despite abundant evidence to show that UCP1 activity is modulated by nucleotides (Klingenberg and Echtay, 2001), FFA and Coenzyme Q (Echtay et al., 2000), no protein that directly modulates UCP1 activity in BAT has been identified to date. Cidea is localized to mitochondria and forms a complex with UCP1. Moreover, coexpression of Cidea and a constitutively active form of UCP1 (UCP1∆3) indicates that Cidea can attenuate the uncoupling activity of UCP1∆3 in yeast cells. It is conceivable that one of the biological functions of Cidea in vivo is to modulate UCP1 activity. As UCP1 is present in great excess in BAT mitochondria, and its in vivo uncoupling activity is much lower than its H+ transport capacity, Cidea inhibition of UCP1 activity may fine tune UCP1 activity and contribute to the 'masking' effect of UCP1 under physiological conditions (Bouillaud et al., 1994; Klingenberg and Echtay, 2001). Alternatively, Cidea inhibition of UCP1 may increase the threshold of UCP1 activity, rendering thermogenesis more sensitive to UCP1 concentration in certain critical ranges. Loss of Cidea inhibition would then result in enhanced uncoupling activity and stimulation of lipolysis, leading to greater energy expenditure, rapid depletion of fat storage and a lean phenotype. Although UCP1 null mice were not obese (possibly owing to compensation by other metabolic processes (Kozak et al., 1991; Liu et al., 2003)), increasing 110 Chapter uncoupling activity by overexpressing UCP1 or UCP3 in transgenic mice clearly prevented obesity (Clapham et al., 2000; Kopecky et al., 1995). Stimulated BAT can convert large amount of calories to heat generation alone. BAT produces heat by oxidation of fatty acids. The fatty acids are combusted in the mitochondria. WAT has few mitochondria and consequently only a limited capacity for β-oxidation of free fatty acids. The excess free fatty acids thus leave the tissue and are transported as a fatty acid-albumin complex to other tissues such as heart, skeletal muscle, liver, kidney and BAT. Uptake and subsequent metabolism of the free fatty acids by these tissues is controlled by the blood concentration of the fatty acids. Two possible routes of metabolism may be followed; β-oxidation or resynthesis of triglycerides for VLDL assembly. The relative flow into each pathway is dependent on the hormonal state. Thus, in most catabolic states, the bulk of the fatty acids are oxidized, but in situations where there is a peripheral resistance to insulin, e.g. diabetes, hepatic synthesis of triglyceride may be significant. It is reported that insulin resistance is correlated with high blood level of free fatty acids (FFA) (Boden, 1998; Scheja et al., 1999). Transport of the activated fatty acid into the mitochondria matrix requires the formation of acylcarnitine on the mitochondrial inner membrane through CPT1 (G.Voet, 1995). This represents the slowest step in the overall oxidation process and is inhibited by malonylcoenzyme A, an intermediate in fatty acid synthesis (G.Voet, 1995) (Figure 8). The amounts of glycerol and FFA released from explants of BAT and WAT that were maintained in vitro were measured to test the lipolysis state of the independent organ. The lower levels of fatty acid released from BAT suggest that Cidea-null mice may have increased fatty acid recycling or fatty acid oxidation. On the contrary, no difference in the release of glycerol or NEFA was observed in WAT from wild type and Cidea-null mice (Figure 30). This gave a hint of how important BAT thermogenesis is in 111 Chapter regulating whole-body homeostasis. It could be speculated that hyperactive BAT serves as a pump to burn out the triglyceride stored in white adipose tissue. It is easy to reason that Cidea may also function by modulating fatty acid metabolism, as Cidea null mice had much lower concentrations of plasma FFA and triglycerides and lower fatty acid release in BAT. It would be very interesting to further investigate Cidea’s localization in the mitochondria and also test whether Cidea deletion could have any effect on the enzyme activities that are critical for β-oxidation of fatty acids. Based on the data presented in Chapter 4, Cidea represents the first protein known in BAT that could modulate UCP1 activity and lipid metabolism and contribute to the development of obesity and diabetes. As Cidea expression is highly restricted to brown adipocytes and its deletion results in increased energy expenditure in BAT without affecting the function of other tissues, Cidea is an ideal target for therapeutic intervention of obesity and type II diabetes. Specifically, drugs that knock down the function of Cidea may be effective in the reversing obesity or alleviating uncontrolled hyperglycemia. In small animals, non-shivering thermogenesis and diet-induced thermogenesis have a great impact on overall body weight, and the question is whether mechanisms to waste energy have evolved also in human energy metabolism. In humans the inability to quantify brown adipose tissue makes it difficult to argue for a role for UCP1 in thermogenesis and energy expenditure (Gonzalez-Barroso et al., 2000). There are data supporting the existence of brown adipocytes and the role of UCP1 in energy dissipation in adult humans (Gonzalez-Barroso et al., 2000). Understanding the mechanisms that regulate the activity of human UCP1 will facilitate understanding of the modulation of energy expenditure in adult humans. Two other CIDE family members, Cideb and FSP27, which share a high sequence similarity with Cidea, have also been identified in mammals. Cideb is expressed at high levels in liver and kidney, whereas 112 Chapter FSP27 is highly expressed in WAT and BAT. Both liver and kidney are important organs involved in fatty acids β-oxidation and glucose metabolism. It will be very interesting to test whether these proteins play similar roles in regulating energy expenditure and the development of obesity and diabetes. The illustration of Cidea’s association with BAT UCP1 and its involvement in fatty acid β-oxidation and lipid metabolism, which eventually affects glucose metabolism and homeostasis regulation, has shedded light on the physiological roles of the CIDE family members in somewhat unexpected ways. 113 Chapter How does CIDE-A disruption result in a lean phenotype? Cold is sensed by the brain Leptin Sympathetic nerves are activated α-adrenergic receptor Ob-Rb Norepinephrine β-adrenegic receptor Adenyl cyclase AMPK AMPKK Activation AMPK-P ACC cAMP ATP HSL ACC-P R2C2 (inactive PKA) 2C (active PKA) R2(cAMP)4 Triglyceride HSL-P (active) FFA Malonyl-CoA Acetyl-CoA Fatty acyl-CoA CPT1 + Brown adipocyte H+ Respiratory chain H H+ ATP Synthase ADP H+ ATP H+ Cidea Fatty acyl-CoA Citric Acid Cycle β-Oxidation Acetyl-CoA H+ Heat UCP1 H+ Figure 36 Schematic diagrams showing how Cidea regulate the BAT metabolism. Based on the novel findings with the Cidea null mice presented here, I propose that Cidea acts as an inhibitor of the uncoupling activity of UCP1 or an inhibitor for 114 Chapter fatty acid transport. As depicted in Figure 36, Cidea probably co-localized with UCP1 at the mitochondria inner membrane, where the two proteins form a complex. Cidea could also play a role in regulating CPT1 activity thus reducing the speed of FFA transport into mitochondria. Since Cidea can negatively regulate UCP1 activity, deficiency of Cidea therefore renders UCP1 hyperactive. Cidea null animals are metabolically inefficient, with energy being wasted as heat and fat storage depleted quickly in response to cold. When mice are housed at room temperature under unstimulated conditions, UCP1 activity is largely inhibited by nucleotide, and the inhibitory effect of Cidea on UCP1 was not obvious. When animals were exposed to cold or stimulated with β-adrenoreceptor analogs, UCP1 activity was increased dramatically due to the loss of inhibition by nucleotides and its stimulation by higher levels of FFA. Loss of Cidea inhibition will then result in hyperactive UCP1 and rapid depletion of triglyceride storage. 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